Fate and effects of microplastic particles in a periphyton-grazer system.

In the aquatic environment, microplastic particles (MP) can accumulate in microbial communities that cover submerged substrata, i.e. in periphyton. Despite periphyton being the essential food source for grazers in the benthic zones, MP transfer from periphyton to benthic biota and its ecotoxicological consequences are unknown. Therefore, in this study, we investigated the effects of 1) MP on embryonal development of freshwater gastropod Physa acuta embryos, 2) MP on adult Physa acuta individuals through dietary exposure and 3) on the MP surface properties. Embryonal development tests were carried out with spherical polyethylene MP in the size of 1-4 µm (MP). Over a period of 28 days, embryonal development and hatching rate were calculated. In the feeding experiments, periphyton was grown in the presence and absence of MP and was then offered to the adult Physa acuta for 42-152 h. The snails readily ingested and subsequently egested MP, together with the periphyton as shown by MP quantification in periphyton, snail soft body tissue and feces. No selective feeding behavior upon MP exposure was detected. The ingestion of MP had no effect on mortality, feeding and defecation rate. Yet, the reproductive output of snails, measured as the number of egg clutches and numbers of eggs per clutch, decreased after the ingestion of MPs, while the hatching success of snail embryos those parents were exposed remained unaffected. In contrast, hatching rate of snail embryos was significantly reduced upon direct MP exposure. MP optical properties were changed upon the incorporation into the periphyton and the passage through the digestive tract. Our results indicate that MP incorporated in periphyton are bioavailable to aquatic grazers, facilitating the introduction of MP into the food chain and having direct adverse effects on the grazers' reproductive fitness.

Organ-specific biotransformation in salmonids: Insight into intrinsic enzyme activity and biotransformation of three micropollutants.

Aquatic ecosystems continue to be threatened by chemical pollution. To what extent organisms are able to cope with chemical exposure depends on their ability to display mechanisms of defense across different organs. Among these mechanisms, biotransformation processes represent key physiological responses that facilitate detoxification and reduce the bioaccumulation potential of chemicals. Biotransformation does not only depend on the ability of different organs to display biotransformation enzymes but also on the affinity of chemicals towards these enzymes. In the present study, we explored the ability of different organs and of two freshwater fish to support biotransformation processes through the determination of in vitro phase I and II biotransformation enzyme activity, and their role in supporting intrinsic clearance and the formation of biotransformation products. Three environmentally relevant pollutants were evaluated: the polycyclic aromatic hydrocarbon (PAH) pyrene (as recommended by the OECD 319b test guideline), the fungicide azoxystrobin, and the pharmaceutical propranolol. Comparative studies using S9 sub-cellular fractions derived from the liver, intestine, gills, and brain of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) revealed significant phase I and II enzyme activity in all organs. However, organ- and species-specific differences were found. In brown trout, significant extrahepatic biotransformation was observed for pyrene but not for azoxystrobin and propranolol. In rainbow trout, the brain appeared to biotransform azoxystrobin. In this same species, propranolol appeared to be biotransformed by the intestine and gills. Biotransformation products could be detected only from hepatic biotransformation, and their profiles and formation rates displayed species-specific patterns and occurred at different magnitudes. Altogether, our findings further contribute to the current understanding of organ-specific biotransformation capacity, beyond the expression and activity of enzymes, and its dependence on specific enzyme-chemical interactions to support mechanisms of defense against exposure.

Bioconcentration Assessment of Three Cationic Surfactants in Permanent Fish Cell Lines.

Cationic surfactants are used in many industrial processes and in consumer products with concurrent release into the aquatic environment, where they may accumulate in aquatic organisms to regulatoryly relevant thresholds. Here, we aimed to better understand the bioconcentration behavior of three selected cationic surfactants, namely N,N-dimethyldecylamine (T10), N-methyldodecylamine (S12), and N,N,N-trimethyltetradecylammonium cation (Q14), in the cells of fish liver (RTL-W1) and gill (RTgill-W1) cell lines. We conducted full mass balances for bioconcentration tests with the cell cultures, in which the medium, the cell surface, the cells themselves, and the plastic compartment were sampled and quantified for each surfactant by HPLC MS/MS. Accumulation in/to cells correlated with the surfactants' alkyl chain lengths and their membrane lipid-water partitioning coefficient, DMLW. Cell-derived bioconcentration factors (BCF) of T10 and S12 were within a factor of 3.5 to in vivo BCF obtained from the literature, while the cell-derived BCF values for Q14 were >100 times higher than the in vivo BCF. From our experiments, rainbow trout cell lines appear as a suitable conservative in vitro screening method for bioconcentration assessment of cationic surfactants and are promising for further testing.

A benchmark dataset for machine learning in ecotoxicology.

The use of machine learning for predicting ecotoxicological outcomes is promising, but underutilized. The curation of data with informative features requires both expertise in machine learning as well as a strong biological and ecotoxicological background, which we consider a barrier of entry for this kind of research. Additionally, model performances can only be compared across studies when the same dataset, cleaning, and splittings were used. Therefore, we provide ADORE, an extensive and well-described dataset on acute aquatic toxicity in three relevant taxonomic groups (fish, crustaceans, and algae). The core dataset describes ecotoxicological experiments and is expanded with phylogenetic and species-specific data on the species as well as chemical properties and molecular representations. Apart from challenging other researchers to try and achieve the best model performances across the whole dataset, we propose specific relevant challenges on subsets of the data and include datasets and splittings corresponding to each of these challenge as well as in-depth characterization and discussion of train-test splitting approaches.

Investigating the bioaccumulation potential of anionic organic compounds using a permanent rainbow trout liver cell line.

Permanent rainbow trout (Oncorhynchus mykiss) cell lines represent potential in vitro alternatives to experiments with fish. We here developed a method to assess the bioaccumulation potential of anionic organic compounds in fish, using the rainbow trout liver-derived RTL-W1 cell line. Based on the availability of high quality in vivo bioconcentration (BCF) and biomagnification (BMF) data and the substances' charge state at physiological pH, four anionic compounds were selected: pentachlorophenol (PCP), diclofenac (DCF), tecloftalam (TT) and benzotriazol-tert-butyl-hydroxyl-phenyl propanoic acid (BHPP). The fish cell line acute toxicity assay (OECD TG249) was used to derive effective concentrations 50 % and non-toxic exposure concentrations to determine exposure concentrations for bioaccumulation experiments. Bioaccumulation experiments were performed over 48 h with a total of six time points, at which cell, medium and plastic fractions were sampled and measured using high resolution tandem mass spectrometry after online solid phase extraction. Observed cell internal concentrations were over-predicted by KOW-derived predictions while pH-dependent octanol-water partitioning (DOW) and membrane lipid-water partitioning (DMLW) gave better predictions of cell internal concentrations. Measured medium and cell internal concentrations at steady state were used to calculate RTL-W1-based BCF, which were compared to DOW- or DMLW-based model approaches and in vivo data. With the exception of PCP, the cell-derived BCF best compared to DOW-based model predictions, which were higher than predictions based on DMLW. All methods predicted the in vivo BCF for diclofenac well. For PCP, the cell-derived BCF was lowest although all BCF predictions underestimated the in vivo BCF by ≥ 1 order of magnitude. The RTL-W1 cells, and all other prediction methods, largely overestimated in vivo BMF, which were available for PCP, TT and BHPP. We conclude that the RTL-W1 cell line can supplement BCF predictions for anionic compounds. For BMF estimations, however, in vitro-in vivo extrapolations need adaptation or a multiple cell line approach.

Addressing chemical pollution in biodiversity research.

Climate change, biodiversity loss, and chemical pollution are planetary-scale emergencies requiring urgent mitigation actions. As these "triple crises" are deeply interlinked, they need to be tackled in an integrative manner. However, while climate change and biodiversity are often studied together, chemical pollution as a global change factor contributing to worldwide biodiversity loss has received much less attention in biodiversity research so far. Here, we review evidence showing that the multifaceted effects of anthropogenic chemicals in the environment are posing a growing threat to biodiversity and ecosystems. Therefore, failure to account for pollution effects may significantly undermine the success of biodiversity protection efforts. We argue that progress in understanding and counteracting the negative impact of chemical pollution on biodiversity requires collective efforts of scientists from different disciplines, including but not limited to ecology, ecotoxicology, and environmental chemistry. Importantly, recent developments in these fields have now enabled comprehensive studies that could efficiently address the manifold interactions between chemicals and ecosystems. Based on their experience with intricate studies of biodiversity, ecologists are well equipped to embrace the additional challenge of chemical complexity through interdisciplinary collaborations. This offers a unique opportunity to jointly advance a seminal frontier in pollution ecology and facilitate the development of innovative solutions for environmental protection.

Bioaccessibility of Organic Compounds Associated with Tire Particles Using a Fish In Vitro Digestive Model: Solubilization Kinetics and Effects of Food Coingestion.

Tire and road wear particles (TRWP) account for an important part of the polymer particles released into the environment. There are scientific knowledge gaps as to the potential bioaccessibility of chemicals associated with TRWP to aquatic organisms. This study investigated the solubilization and bioaccessibility of seven of the most widely used tire-associated organic chemicals and four of their degradation products from cryogenically milled tire tread (CMTT) into fish digestive fluids using an in vitro digestion model based on Oncorhynchus mykiss. Our results showed that 0.06-44.1% of the selected compounds were rapidly solubilized into simulated gastric and intestinal fluids within a typical gut transit time for fish (3 h in gastric and 24 h in intestinal fluids). The environmentally realistic scenario of coingestion of CMTT and fish prey was explored using ground Gammarus pulex. Coingestion caused compound-specific changes in solubilization, either increasing or decreasing the compounds' bioaccessibility in simulated gut fluids compared to CMTT alone. Our results emphasize that tire-associated compounds become accessible in a digestive milieu and should be studied further with respect to their bioaccumulation and toxicological effects upon passage of intestinal epithelial cells.

Wastewater microorganisms impact microbial diversity and important ecological functions of stream periphyton.

Effluents of wastewater treatment plants can impact microbial communities in the receiving streams. However, little is known about the role of microorganisms in wastewater as opposed to other wastewater constituents, such as nutrients and micropollutants. We aimed therefore at determining the impact of wastewater microorganisms on the microbial diversity and function of periphyton, key microbial communities in streams. We used a flow-through channel system to grow periphyton upon exposure to a mixture of stream water and unfiltered or ultra-filtered wastewater. Impacts were assessed on periphyton biomass, activities and tolerance to micropollutants, as well as on microbial diversity. Our results showed that wastewater microorganisms colonized periphyton and modified its community composition, resulting for instance in an increased abundance of Chloroflexi and a decreased abundance of diatoms and green algae. This led to shifts towards heterotrophy, as suggested by the changes in nutrient stoichiometry and the increased mineralization potential of carbon substrates. An increased tolerance towards micropollutants was only found for periphyton exposed to unfiltered wastewater but not to ultra-filtered wastewater, suggesting that wastewater microorganisms were responsible for this increased tolerance. Overall, our results highlight the need to consider the role of wastewater microorganisms when studying potential impacts of wastewater on the receiving water body.

Comparative study of the sensitivity of two freshwater gastropods, Lymnaea stagnalis and Planorbarius corneus, to silver nanoparticles: bioaccumulation and toxicity.

Metal-based nanoparticles (NPs) are considered detrimental to aquatic organisms due to their potential accumulation. However, little is known about the mechanisms underlying these effects and their species-specificity. Here we used stable silver (Ag) NPs (20 nm, from 10 to 500 µg/L) with a low dissolution rate (≤2.4%) to study the bioaccumulation and biological impacts in two freshwater gastropods: Lymnaea stagnalis and Planorbarius corneus. No mortality was detected during the experiments. Ag bioaccumulation showed a dose-related increase with an enhanced concentration in both species after 7d exposure. L. stagnalis displayed a higher accumulation for AgNPs than P. corneus (e.g., up to 18- and 15-fold in hepatopancreas and hemolymph, respectively) which could be due to the more active L. stagnalis having greater contact with suspended AgNPs. Furthermore, the hepatopancreas and stomach were preferred organs for bioaccumulation compared to the kidney, mantle and foot. Regarding biological responses, the hemolymph rather than hepatopancreas appeared more susceptible to oxidative stress elicited by AgNPs, as shown by significantly increasing lipid peroxidation (i.e., formation of malondialdehyde). Neurotoxicity was detected in L. stagnalis when exposed to high concentrations (500 µg/L). Comparison with impacts elicited by dissolved Ag revealed that the effects observed on AgNPs exposure were mainly attributable to NPs. These results highlighted the relationship between the physiological traits, bioaccumulation, and toxicity responses of these two species to AgNPs and demonstrated the necessity of species-specificity considerations when assessing the toxicity of NPs.

Predicting chemical hazard across taxa through machine learning.

We applied machine learning methods to predict chemical hazards focusing on fish acute toxicity across taxa. We analyzed the relevance of taxonomy and experimental setup, showing that taking them into account can lead to considerable improvements in the classification performance. We quantified the gain obtained throught the introduction of taxonomic and experimental information, compared to classification based on chemical information alone. We used our approach with standard machine learning models (K-nearest neighbors, random forests and deep neural networks), as well as the recently proposed Read-Across Structure Activity Relationship (RASAR) models, which were very successful in predicting chemical hazards to mammals based on chemical similarity. We were able to obtain accuracies of over 93% on datasets where, due to noise in the data, the maximum achievable accuracy was expected to be below 96%. The best performances were obtained by random forests and RASAR models. We analyzed metrics to compare our results with animal test reproducibility, and despite most of our models "outperform animal test reproducibility" as measured through recently proposed metrics, we showed that the comparison between machine learning performance and animal test reproducibility should be addressed with particular care. While we focused on fish mortality, our approach, provided that the right data is available, is valid for any combination of chemicals, effects and taxa.

In Vitro Digestion of Tire Particles in a Fish Model (Oncorhynchus mykiss): Solubilization Kinetics of Heavy Metals and Effects of Food Coingestion.

Tire and road wear particles (TRWP) have been shown to represent a large part of anthropogenic particles released into the environment. Nevertheless, the potential ecological risk of TRWP in the different environmental compartments and their potential toxic impacts on terrestrial and aquatic organisms remain largely underinvestigated. Several heavy metals compose TRWP, including Zn, which is used as a catalyst during the vulcanization process of rubber. This study investigated the solubilization potential of metals from cryogenically milled tire tread (CMTT) and TRWP in simulated gastric fluids (SFGASTRIC) and simulated intestinal fluids (SFINTESTINAL) designed to mimic rainbow trout (Oncorhynchus mykiss) gastrointestinal conditions. Our results indicate that the solubilization of heavy metals was greatly enhanced by gastrointestinal fluids compared to that by mineral water. After a 26 h in vitro digestion, 9.6 and 23.0% of total Zn content of CMTT and TRWP, respectively, were solubilized into the simulated gastrointestinal fluids. Coingestion of tire particles (performed with CMTT only) and surrogate prey items (Gammarus pulex) demonstrated that the animal organic matter reduced the amount of bioavailable Zn solubilized from CMTT. Contrastingly, in the coingestion scenario with vegetal organic matter (Lemna minor), high quantities of Zn were solubilized from L. minor and cumulated with Zn solubilized from CMTT.

Characterization of the ERK1/2 phosphorylation profile in human and fish liver cells upon exposure to chemicals of environmental concern.

We developed phospho-ERK1/2 ELISA for human and rainbow trout liver cells, employing HepG2 and RTL-W1 cell lines as models. The assay was applied to detect changes in ERK1/2 activity for nine chemicals, added over a wide concentration range and time points. Cell viability was measured to separate ERK1/2 regulation from cytotoxicity. Perfluorooctane sulfonate and carbendazim did not change ERK1/2 activity; influence on ERK1/2 due to cytotoxicity was indicated for tributyltin and cypermethrin. Mancozeb, benzo[a]pyrene, and bisphenol A stimulated ERK1/2 up to ∼2- (HepG2) and 1.5 (RTL-W1)-fold, though the kinetics differed between chemicals and cell lines. Bisphenol A and benzo[a]pyrene were the most potent concentration-wise, altering ERK1/2 activity in pM (HepG2) to nM (RTL-W1) range. While atrazine and ibuprofen increased ERK1/2 activity by ∼2-fold in HepG2, they did not initiate an appreciable response in RTL-W1. This assay proved to be a sensitive, medium- to high-throughput tool for detecting unrecognized ERK1/2-disrupting chemicals.

Toxic effects of substituted p-benzoquinones and hydroquinones in in vitro bioassays are altered by reactions with the cell assay medium.

Substituted para-benzoquinones and hydroquinones are ubiquitous transformation products that arise during oxidative water treatment of phenolic precursors, for example through ozonation or chlorination. The benzoquinone structural motive is associated with mutagenicity and carcinogenicity, and also with induction of the oxidative stress response through the Nrf2 pathway. For either endpoint, toxicological data for differently substituted compounds are scarce. In this study, oxidative stress response, as indicated by the AREc32 in vitro bioassay, was induced by differently substituted para-benzoquinones, but also by the corresponding hydroquinones. Bioassays that indicate defense against genotoxicity (p53RE-bla) and DNA repair activity (UmuC) were not activated by these compounds. Stability tests conducted under incubation conditions, but in the absence of cell lines, showed that tested para-benzoquinones reacted rapidly with constituents of the incubation medium. Compounds were abated already in phosphate buffer, but even faster in biological media, with reactions attributed to amino- and thiol-groups of peptides, proteins, and free amino acids. The products of these reactions were often the corresponding substituted hydroquinones. Conversely, differently substituted hydroquinones were quantitatively oxidized to p-benzoquinones over the course of the incubation. The observed induction of the oxidative stress response was attributed to hydroquinones that are presumably oxidized to benzoquinones inside the cells. Despite the instability of the tested compounds in the incubation medium, the AREc32 in vitro bioassay could be used as an unspecific sum parameter to detect para-benzoquinones and hydroquinones in oxidatively treated waters.

Impact of wastewater on the microbial diversity of periphyton and its tolerance to micropollutants in an engineered flow-through channel system.

Wastewater treatment plants (WWTPs) play an important role in retaining organic matter and nutrients but to a lesser extent micropollutants. Therefore, treated wastewater is recognized as a major source of multiple stressors, including complex mixtures of micropollutants. These can potentially affect microbial communities in the receiving water bodies and the ecological functions they provide. In this study, we evaluated in flow-through channels the consequences of an exposure to a mixture of stream water and different percentages of urban WWTP effluent, ranging from 0% to 80%, on the microbial diversity and function of periphyton communities. Assuming that micropollutants exert a selective pressure for tolerant microorganisms within communities, we further examined the periphyton sensitivity to a micropollutant mixture extracted from passive samplers that were immersed in the wastewater effluent. As well, micropollutants in water and in periphyton were comprehensively quantified. Our results show that micropollutants detected in periphyton differed from those found in water, both in term of concentration and composition. Especially photosystem II inhibitors accumulated in periphyton more than other pesticides. Although effects of other substances cannot be excluded, this accumulation may have contributed to the observed higher tolerance of phototrophic communities to micropollutants upon exposure to 30% and 80% of wastewater. On the contrary, no difference in tolerance was observed for heterotrophic communities. Exposure to the gradient of wastewater led to structural differences in both prokaryotic and eukaryotic communities. For instance, the relative abundance of cyanobacteria was higher with increasing percentage of wastewater effluent, whereas the opposite was observed for diatoms. Such results could indicate that differences in community structure do not necessarily lead to higher tolerance. This highlights the need to consider other wastewater constituents such as nutrients and wastewater-derived microorganisms that can modulate community structure and tolerance. By using engineered flow-through channels that mimic to some extent the required field conditions for the development of tolerance in periphyton, our study constitutes a base to investigate the mechanisms underlying the increased tolerance, such as the potential role of microorganisms originating from wastewater effluents, and different treatment options to reduce the micropollutant load in effluents.

New Stable Cell Lines Derived from the Proximal and Distal Intestine of Rainbow Trout (Oncorhynchus mykiss) Retain Several Properties Observed In Vivo.

We derived two novel cell lines from rainbow trout (RT) proximal (RTpi-MI) and distal intestine (RTdi-MI) and compared them with the previously established continuous cell line RTgutGC. Intestinal stem cells, differentiating and differentiated epithelial cells, and connective cells were found in all cell lines. The cell lines formed a polarized barrier, which was not permeable to large molecules and absorbed proline and glucose. High seeding density induced their differentiation into more mature phenotypes, as indicated by the downregulation of intestinal stem cell-related genes (i.e., sox9, hopx and lgr5), whereas alkaline phosphatase activity was upregulated. Other enterocyte markers (i.e., sglt1 and pept1), however, were not regulated as expected. In all cell lines, the presence of a mixed population of epithelial and stromal cells was characterized for the first time. The expression by the stromal component of lgr5, a stem cell niche regulatory molecule, may explain why these lines proliferate stably in vitro. Although most parameters were conserved among the three cell lines, some significant differences were observed, suggesting that characteristics typical of each tract are partly conserved in vitro as well.

Cytotoxicity, Accumulation and Translocation of Silver and Silver Sulfide Nanoparticles in contact with Rainbow Trout Intestinal Cells.

Silver nanoparticles (Ag NPs) are widely used in consumer products especially because of their antimicrobial properties. However, this wide usage of Ag NPs is accompanied by their release into the environment where they will be rapidly transformed to other silver species - especially silver sulfide (Ag2S). In the present study, we synthesized Ag NPs and sulfidized them to obtain a core-shell system Ag@Ag2S NPs. Both types of particles form stable dispersions with hydrodynamic diameters of less than 100 nm when diluted in water, but tend to form micrometer-sized agglomerates in biological exposure media. Application of Ag and Ag@Ag2S NPs to rainbow trout intestinal cells (RTgutGC) resulted in a concentration-dependent cytotoxicity for both types of particles, as assessed by a three-endpoint assay for metabolic activity, membrane integrity and lysosomal integrity. The Ag NPs were shown to be slightly more toxic than the Ag@Ag2S NPs. Adding Ag or Ag@Ag2S NPs to RTgutGC cells, grown on a permeable membrane to mimic the intestinal barrier, revealed considerable accumulation of silver for both types of particles. Indeed, the cells significantly attenuated the NP translocation, allowing only a fraction of the metal to translocate across the intestinal epithelium. These findings support the notion that the intestine constitutes an important sink for Ag NPs and that, despite the reduced cytotoxicity of a sulfidized NP form, the particles can enter fish where they may constitute a long-term source for silver ion release and cytotoxicity.

A ribonucleoprotein transfection strategy for CRISPR/Cas9-mediated gene editing and single cell cloning in rainbow trout cells.

BACKGROUND: The advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology marked the beginning of a new era in the field of molecular biology, allowing the efficient and precise creation of targeted mutations in the genome of every living cell. Since its discovery, different gene editing approaches based on the CRISPR/Cas9 technology have been widely established in mammalian cell lines, while limited knowledge is available on genetic manipulation in fish cell lines. In this work, we developed a strategy to CRISPR/Cas9 gene edit rainbow trout (Oncorhynchus mykiss) cell lines and to generate single cell clone-derived knock-out cell lines, focusing on the phase I biotransformation enzyme encoding gene, cyp1a1, and on the intestinal cell line, RTgutGC, as example. RESULTS: Ribonucleoprotein (RNP) complexes, consisting of the Cas9 protein and a fluorescently labeled crRNA/tracrRNA duplex targeting the cyp1a1 gene, were delivered via electroporation. A T7 endonuclease I (T7EI) assay was performed on flow cytometry enriched transfected cells in order to detect CRISPR-mediated targeted mutations in the cyp1a1 locus, revealing an overall gene editing efficiency of 39%. Sanger sequencing coupled with bioinformatic analysis led to the detection of multiple insertions and deletions of variable lengths in the cyp1a1 region directed by CRISPR/Cas9 machinery. Clonal isolation based on the use of cloning cylinders was applied, allowing to overcome the genetic heterogeneity created by the CRISPR/Cas9 gene editing. Using this method, two monoclonal CRISPR edited rainbow trout cell lines were established for the first time. Sequencing analysis of the mutant clones confirmed the disruption of the cyp1a1 gene open reading frame through the insertion of 101 or 1 base pair, respectively. CONCLUSIONS: The designed RNP-based CRISPR/Cas9 approach, starting from overcoming limitations of transfection to achieving a clonal cell line, sets the stage for exploiting permanent gene editing in rainbow trout, and potentially other fish cells, for unprecedented exploration of gene function.

Zebrafish early life stages as alternative model to study 'designer drugs': Concordance with mammals in response to opioids.

The number of new psychoactive substances (NPS) on the illicit drug market increases fast, posing a need to urgently understand their toxicity and behavioural effects. However, with currently available rodent models, NPS assessment is limited to a few substances per year. Therefore, zebrafish (Danio rerio) embryos and larvae have been suggested as an alternative model that would require less time and resources to perform an initial assessment and could help to prioritize substances for subsequent evaluation in rodents. To validate this model, more information on the concordance of zebrafish larvae and mammalian responses to specific classes of NPS is needed. Here, we studied toxicity and behavioural effects of opioids in zebrafish early life stages. Synthetic opioids are a class of NPS that are often used in pain medication but also frequently abused, having caused multiple intoxications and fatalities recently. Our data shows that fentanyl derivatives were the most toxic among the tested opioids, with toxicity in the zebrafish embryo toxicity test decreasing in the following order: butyrfentanyl>3-methylfentanyl>fentanyl>tramadol> O-desmethyltramadol>morphine. Similar to rodents, tramadol as well as fentanyl and its derivatives led to hypoactive behaviour in zebrafish larvae, with 3-methylfentanyl being the most potent. Physico-chemical properties-based predictions of chemicals' uptake into zebrafish embryos and larvae correlated well with the effects observed. Further, the biotransformation pattern of butyrfentanyl in zebrafish larvae was reminiscent of that in humans. Comparison of toxicity and behavioural responses to opioids in zebrafish and rodents supports zebrafish as a suitable alternative model for rapidly testing synthetic opioids.